The long range objective of our studies is to determine the biological significance of amyloid-type protein aggregation and mechanisms for its control in the epididymal lumen using the cystatins as molecular models. The abnormal accumulation of aggregated protein, also known as amyloid, is common in degenerative diseases including Alzheimer's disease. Amyloid in the testis and epididymis has also been implicated in human infertility. Proteins, including the cystatins, which can self-aggregate and form amyloid adopt a common cytotoxic structure during their aggregation. Because of the active secretion of proteins and profound removal of fluid by the epithelium, macromolecular crowding is likely to occur in the tubular lumen of the epididymis causing amyloid-type protein aggregation. However, because of its critical role in sperm maturation, surveillance/clearance mechanisms must be in place to control this process and prevent a pathological accumulation of cytotoxic aggregates. We have established that the cystatins CRES and cystatin C are present in the caput lumen as high molecular mass oligomeric complexes. We have also shown that CRES is associated with defined structures in the epididymal lumen. Furthermore, in vitro CRES and cystatin C form soluble amyloid precursors, which may be cytotoxic, as well as amyloid fibrils. We have also determined that male mice expressing the mutant L68Q cystatin C, an unstable and highly amyloidogenic form, are infertile possibly due to excess cystatin C oligomeric complexes in the lumen. These novel findings emphasize the critical nature of controlling protein aggregation in the epididymis. One mechanism by which the epididymis may control aggregation is by transglutaminase (TG) crosslinking resulting in protein aggregates in a nontoxic conformation. In support we have shown TG activity in the lumen, that CRES is a substrate for TG, and that TG will form CRES oligomers in caput fluid. Based on these studies we propose that amyloid-type protein aggregation occurs in the epididymal lumen and that quality control mechanisms, such as TG crosslinking, prevent the accumulation of toxic protein aggregates thereby maintaining normal epididymal function. We also propose that conditions that impair these protective mechanisms can negatively impact sperm maturation and function. We will address this hypothesis by: 1) characterizing amyloid-type aggregation in the epididymal lumen; 2) examine the pathological consequences of excessive amyloid aggregation; and 3) examine mechanisms of extracellular quality control in the epididymis. Narrative The objective of our studies is to determine the biological significance of amyloid-type protein aggregation and mechanisms for its control in the epididymal lumen using the cystatins as molecular models. A completion of our aims will provide valuable information for our understanding of amyloid formation not only in the reproductive tract and its potential role in infertility but in general and as such may lead to new therapies and/or markers for diseases associated with extracellular aggregated proteins such as Alzheimer's disease.